Method for monitoring environmental states of a microscope sample with an electron microscope sample holder

ABSTRACT

An apparatus and a method for measuring and monitoring the properties of a fluid, for example, pressure, temperature, and chemical properties, within a sample holder for an electron microscope. The apparatus includes at least one fiber optic sensor used for measuring temperature and/or pressure and/or pH positioned in proximity of the sample.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of earlier-filed U.S. applicationSer. No. 14/626,234, filed Feb. 19, 2015, titled “METHOD FOR MONITORINGENVIRONMENTAL STATES OF A MICROSCOPE SAMPLE WITH AN ELECTRON MICROSCOPESAMPLE HOLDER.” This application and U.S. application Ser. No.14/626,234 claim the benefit of priority of U.S. provisional applicationNo. 61/941,743, filed on Feb. 19, 2014. U.S. application Ser. No.14/626,234 and U.S. provisional application No. 61/941,743 areincorporated herein by this reference.

TECHNICAL FIELD

The invention relates generally to a method for measuring and monitoringthe properties of a fluid such as a gas or liquid within a sample holderfor an electron microscope. e.g., a transmission electron microscope(TEM), a scanning transmission electron microscopy (STEM), andvariations of the scanning electron microscopes (SEM) that usetraditional TEM-type holders and stages, for imaging and analysis.

BACKGROUND

The sample holder is a component of an electron microscope providing thephysical support for samples under observation. Sample holderstraditionally used for TEMs and STEMs, as well as some modern SEMs,consist of a rod that is comprised of three key regions: the end, thebarrel and the specimen tip. In addition to supporting the sample, thesample holder provides an interface between the inside of the instrument(i.e., a vacuum environment) and the outside world.

To use the sample holder, one or more samples are first placed on asample support device. The sample support device is then mechanicallyfixed in place at the specimen tip, and the sample holder is insertedinto the electron microscope through a load-lock. During insertion, thesample holder is pushed into the electron microscope, assisted by thevacuum within the microscope, until it stops, which results in thespecimen tip of the sample holder being located in the column of themicroscope. At this point, the barrel of the sample holder bridges thespace between the inside of the microscope and the outside of the loadlock, and the end of the sample holder is outside the microscope. Tomaintain an ultra-high vacuum environment inside the electronmicroscope, flexible o-rings are typically found along the barrel of thesample holder, and these o-rings seal against the microscope when thesample holder is inserted. The exact shape and size of the sample holdervaries with the type and manufacturer of the electron microscope, buteach holder contains these three key regions.

The sample holder can also be used to provide stimulus to the sample,and this stimulus can include temperature (e.g., heating or cooling),electricity (e.g., applying a voltage or current), mechanical (e.g.,applying stress or strain), gas or liquid (e.g., containing a sample ina specific gaseous or liquid environment), or more than one of these atonce. For example, a syringe pump can be used to force liquids through asample holder containing a sample during imaging. This equipment isoutside of the microscope, and various connectors are used to bring thisstimulus down the length of the holder to the sample holder, and to thesamples. For example, microfluidic tubing can be used to supply liquidsfrom the syringe pump to the sample.

One configuration is an environmental cell wherein two semiconductordevices comprising thin windows are used, and samples are sandwichedbetween the two semiconductor devices, and the environment in proximityof the sample, including an electrical field, temperature, and a gas orliquid flow, can be precisely controlled. The present inventorspreviously described apparatuses and methods to contact and aligndevices used to form liquid or gas cells in International PatentApplication No. PCT/US2011/46282 filed on Aug. 2, 2011 entitled“ELECTRON MICROSCOPE SAMPLE HOLDER FOR FORMING A GAS OR LIQUID CELL WITHTWO SEMICONDUCTOR DEVICES,” which is hereby incorporated herein in itsentirety.

It is advantageous to be able to monitor the conditions of theenvironment at or near the sample. Conditions of particular interestinclude temperature, pressure and chemical properties such as pH.Disadvantageously, most traditional sensors for measuring suchconditions are too large to incorporate into the relatively small spacesof an electron microscope holder. Although some traditional sensors willallow for the measurements farther away from the sample, this limits theability to ensure high accuracy and dynamic control. A sensor mountedvery close to the sample of the holder is needed. One technology thatcould allow for localized measurements of environmental properties insmall areas is fiber optic sensors. Since some of these sensors areavailable in diameters less than 150 micrometers, they are small enoughto overcome previous challenges and by using a delicate balance ofdesign features and parameters, there are a variety of unique ways thesesensors can be assembled into the holders.

Fiber optic sensors operate by transporting light by wavelength orintensity to provide information about the environment surrounding thesensor. The environment surrounding a fiber optic sensor is usuallyliquid or gas. Fiber optic sensors can be categorized as intrinsic orextrinsic. Extrinsic fiber optic sensors simply use an optical fiber totransport light. An example is the laser induced fluorescence (LIF) conepenetrometer. The optical fiber is only a conduit for the laser inducedfluorescence to be transported to an uphole detector. In contrast,intrinsic fiber optic sensors use the fiber directly as the detector.

There are a variety of intrinsic fiber optic sensors that could be usedto measure environmental properties at the tip of an environmental TEMholder. These include pressure, temperature and chemical fiber opticsensors.

There are specific advantages to measuring temperature of the gas orliquid environment. One primary advantage is accuracy. By monitoring thetemperature of the gas or liquid within close proximity to themicroscope sample, heat transfer losses can be minimized by ensuringthat the temperature is readjusted in real time to the precise requiredtemperature, which is particularly important if the heat source islocated at a relative distance from the sample. Many experiments requirean accurate temperature to conduct a successful experiment. For example,live biological samples will die if the liquid temperature is too highand certain electrochemical reactions require a stable temperature.

Measuring the absolute pressure of the gas or liquid at the sampleoffers many advantages as well. One advantage is safety. By monitoringand controlling the pressure, the closed cell system can be kept at asafe level, e.g., avoiding overpressure which can break thinsemiconductor membranes and introduce gas into the column of theelectron microscope. A second advantage is the ability to calculate thetemperature within a gas environment. Since the actual temperature ofany given gas or gas mixture is a function of the pressure, and becauseconvective heat transfer of gas can cool heated objects placed in agaseous environment as a function of pressure, the gas pressure can bemeasured to accurately calculate the temperature of the heated object.Thirdly, the reaction rate of many reactions is pressure dependent, andthe ability to measure and control the pressure is essential tounderstanding and analyzing experimental results.

Monitoring chemical properties at the sample, such as pH levels in aliquid environmental cell, enable the user to correlate the value ofthis property in relationship to a reaction(s) observation. It alsoallows the user or the system to make adjustments to the composition ormetering rate of the fluid flowing to the sample region.

It is therefore an object of the present invention to provide a sampleholder and a sensor or sensors to allow the user to accurately andefficiently measure the environmental properties including pressure,temperature and pH on or within the electron microscope holder inproximity of the sample.

SUMMARY

The present invention generally relates to electron microscope sampleholders comprising intrinsic fiber optic pressure, temperature and pHmeasurement devices, methods for measuring the pressure, temperature andpH in proximity of the sample in the sample holder, and uses of thesample holders.

In one aspect, an electron microscope sample holder is described, saidsample holder comprising an end, a barrel, a specimen tip, and at leastone fiber optic sensor used for measuring temperature and/or pressureand/or pH, wherein the specimen tip comprises a closed cell comprising afluidic reservoir, wherein said sensor(s) extends from the end of thesample holder along the barrel to the fluidic reservoir of the closedcell to measure the temperature and/or pressure and/or pH of the fluidicreservoir.

In another aspect, another electron microscope sample holder isdescribed, said sample holder comprising an end, a barrel, a specimentip, and at least one fiber optic sensor used for measuring temperatureand/or pressure, wherein a sample positioned in the specimen tip isexposed to gases and pressures established in an environmental electronmicroscope, wherein said sensor extends from the end of the sampleholder along the barrel to an area at or near the sample.

In still another aspect, a method of measuring the pressure and/ortemperature and/or pH in proximity of a sample in a closed cell isdescribed, said method comprising positioning a sample in the fluidicreservoir of the sample holder, and measuring the pressure and/ortemperature and/or pH using said sensor(s), wherein the sample holdercomprises an end, a barrel, a specimen tip, and at least one fiber opticsensor used for measuring temperature and/or pressure and/or pH, whereinthe specimen tip comprises a closed cell comprising a fluidic reservoir,wherein said sensor(s) extends from the end of the sample holder alongthe barrel to the fluidic reservoir of the closed cell to measure thetemperature and/or pressure and/or pH of the fluidic reservoir.

In yet another embodiment, a method of measuring the pressure and/ortemperature and/or pH in proximity of a sample in an environmentalelectron microscope is described, said method comprising positioning asample in on a sample support device in the sample holder, and measuringthe pressure and/or temperature and/or pH using said sensor(s), whereinthe sample holder comprises an end, a barrel, a specimen tip, and atleast one fiber optic sensor used for measuring temperature and/orpressure, wherein a sample positioned in the specimen tip is exposed togases and pressures established in an environmental electron microscope,wherein said sensor extends from the end of the sample holder along thebarrel to an area at or near the sample.

Other aspects, features and embodiments of the invention will be morefully apparent from the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the general sample holder described herein.

FIG. 2A is a plan view of an embodiment of the closed sample holder cellwith the sample holder cover on.

FIG. 2B is a plan view of the sample holder cell of FIG. 2A with thesample holder cover off.

FIG. 2C is a plan view of the sample holder cell of FIG. 2B with thelarge MEMS chip removed.

FIG. 2D is a plan view of the sample holder cell of FIG. 2C with thesmall MEMS chip removed.

FIG. 3 is a plan view of the sample holder cell illustrating theplacement of the fluidic tubing relative to the fluidic reservoir.

FIG. 4A is a plan view of the sample holder illustrating an embodimentof the placement of the fiber optic sensor assembly.

FIG. 4B illustrates examples of the fiber optic sensor assemblies.

FIG. 5 is a plan view of the sample holder cell illustrating analternative placement of a pressure or temperature or pH sensorassembly.

FIG. 6 is a plan view of the sample holder cell illustrating anotheralternative placement of a pressure or temperature or pH sensorassembly.

FIG. 7 is a plan view of the sample holder cell illustrating anotheralternative placement of the fiber optic sensor assembly, wherein theend of the sensor is positioned within a fluidic reservoir along one ofthe fluidic ports.

FIG. 8 is a plan view of the non-closed sample holder cell illustratingthe use of pressure or temperature sensors on non-closed cell electronmicroscope holders.

DETAILED DESCRIPTIONS

The present invention generally relates to sample holders comprisingintrinsic fiber optic pressure, temperature and pH measurement devices,methods for measuring the pressure, temperature and pH in proximity ofthe sample in the sample holder, and uses of the sample holders. It isto be understood that the sample holder and sample holder interfacedescribed herein are compatible with and may be interfaced with thesample support devices, e.g., semiconductor sample support devices,disclosed in International Patent Application Nos. PCT/US08/63200 filedon May 9, 2008, PCT/US11/46282 filed on Aug. 2, 2011, and PCT/US08/88052filed on Dec. 22, 2008, which are all incorporated herein by referencein their entireties. It should also be appreciated by one skilled in theart that alternative sample support devices may be interfaced with thesample holder described herein. The sample holder provides mechanicalsupport for one or more samples or sample support devices and may alsoprovide other stimuli (e.g., temperature, electricity, mechanical,chemical, gas or liquid, or any combination thereof) to the samples orsample support devices. The sample holder can be manufactured with tips,barrels and ends of various shapes and sizes such that the sample holderfits any manufacturer's electron microscope.

As used herein, a “sample support device” corresponds to a structurethat holds a sample for microscopic imaging. A sample support device canprovide an experimental region. Devices may include one, more than oneor even an array of experimental regions and may include integratedfeatures such as electrodes, thermocouples, and/or calibration sites, asreadily determined by one skilled in the art. One preferred embodimentincludes sample support devices made with MEMS technology and with thinmembrane regions (continuous or perforated) for supporting a sample inthe experimental region. Examples of sample support devices includewindow devices, thermal devices and electrochemical devices. When thesample holder accommodates two sample support devices, typically one isa window device and one is a thermal device or an electrochemicaldevice.

As defined herein, a “membrane region” on the sample support devicecorresponds to unsupported material comprising, consisting of, orconsisting essentially of carbon, silicon nitride, SiC or other thinfilms generally 1 micron or less having a low tensile stress (<500 MPa),and providing a region at least partially electron transparent regionfor supporting the at least one sample. The membrane region may includeholes or be hole-free. The membrane region may be comprised of a singlematerial or a layer of more than one material and may be eitheruniformly flat or contain regions with varying thicknesses. The membraneregion is generally supported by a thicker frame.

The general area of “in situ” electron microscopy involves applyingstimulus to a sample during imaging. The stimulus could be thermal(heating or cooling), electrical (applying a voltage or current),mechanical (applying stress or strain), chemical (containing a sample ina specific chemical environment), or several of these at once.

The sample holder of the present disclosure is broadly illustrated inFIG. 1, wherein the sample holder includes tubing inside the electronmicroscope (EM) holder that travels to and from the closed cell at thespecimen tip. The placement of the tubing is just for generalillustration and is not intended to limit the holder in any way. Thetubing permits fluids, e.g., gases or liquids, to travel to the closedcell, for in situ analysis of the sample positioned in the closed cell.

FIGS. 2A-2D illustrate an example of the closed cell that is positionedat the specimen tip. The closed cell in FIGS. 2A-2D is just for generalillustration and is not intended to limit the closed cell in any way.FIG. 2A is a plan view of a general closed cell, wherein a cover of theclosed cell cover is shown positioned and affixed, e.g., with screws, tothe cell. FIG. 2B is a plan view of the closed cell with the cover off,revealing the first of two MEMS chips (i.e., a sample support device)positioned in the cell. FIG. 2C is a plan view of the closed cellshowing the second of two MEMS chips after the first MEMS chip isremoved. The large and small MEMS chips are stacked on top of oneanother and the sample “sandwiched” between the two chips. FIG. 2C alsoreveals the first of two O-rings, which is positioned below the largeMEMS chip to seal the cell so liquid or gas can be introduced into thecell. FIG. 2D is a plan view of the closed cell showing the bottom ofthe cell after the second MEMS chip is removed. FIG. 2D also reveals thesecond of two O-rings, which is positioned below the small MEMS chip toform the second seal so liquid or gas can be introduced into the cell.The fluidic reservoir in FIG. 2D corresponds to the area between the twoO-rings when the MEMS chips are in place. Although not illustrated inFIGS. 2A-2D per se, the fluidic reservoir indicated in FIG. 2D has depthto accommodate the large and small MEMS chips. It should be appreciatedthat the “closed cell” remains in fluid communication with fluidicinlets and hence the closed cell receives fluids from an external sourceand fluids are returned from the closed cell to an external source.

FIG. 3 illustrates the placement of the fluidic tubing at the fluidicreservoir, wherein the end of the tubing is positioned in the fluidicreservoir.

FIG. 4A illustrates the placement of the fiber optic sensor assembly(i.e., pressure or temperature or chemical assembly) according to afirst embodiment of the invention. In FIG. 4A, the end of the sensor ispositioned in the fluidic reservoir at or near the sample and sealed.Examples of the fiber optic sensor assemblies are shown in FIG. 4B,e.g., from FISO Technologies, wherein the fiber optic sensor extendsthrough the sample holder and eventually to a control interface.

Mainly three technologies are presently commercially available forpressure measurement with fiber-optic sensors: intensity-based, fiberBragg gratings and Fabry-Perot technology. Fabry-Perot (F-P) technologymay be the best compromise in terms of pressure ranges, high sensitivityand miniature size. In F-P pressure sensors, a reflective membrane isassembled above a vacuumed cavity with a semi-reflective layer at thebottom forming a F-P cavity that changes in length with the pressurechanges that deflect the membrane. The interference pattern created bythe F-P cavity can be used to measure precisely the diaphragm deflectionand thus the pressure changes.

For a chemical fiber optic sensor, a portion of the optical fibercladding is removed and replaced with a chemically selective layer. Thesensor is then placed directly into the media to be analyzed.Interaction of the analyte with the chemically selective layer creates achange in absorbance, reflectance, fluorescence, or light polarization.The optical change is then detected by measuring changes in the lightcharacteristic carried by the optical fiber.

Intrinsic temperature sensors with a wide measurement range typicallyuse an interferometric sensing method. These interferometric sensorswork by sending a light through a reference fiber and also a sensingfiber. As the temperature changes the physical dimensions of the sensingfiber, there would then be a phase difference in between the light whileit travels between the two fibers previously stated. That phasedifference can be measured by transforming into a physical dimensionchange, and lastly it will give the temperature information needed.

FIG. 5 illustrates an alternative placement of a pressure or temperatureor pH sensor assembly according to a second embodiment of the invention.In FIG. 5, the end of the sensor is positioned in the fluidic reservoirin proximity to a fluidic ingress or egress and sealed. For example, thesensor can be positioned at the fluidic ingress. Alternatively, thesensor can be positioned at the fluidic egress. An example of the sensoris the fiber optic cable of FIG. 4B, wherein the fiber optic sensorextends through the sample holder and eventually to a control interface.

FIG. 6 illustrates another alternative placement of the pressure ortemperature or pH sensor assembly according to a third embodiment of theinvention. In FIG. 6, the end of the sensor is positioned in the fluidicreservoir in proximity to both the fluidic ingress and egress andsealed. An additional fluidic tubing pathway is illustrated as well,although that is optional. Examples of the fiber optic sensor assembliesare shown in FIG. 4B, wherein the fiber optic sensor extends through thesample holder and eventually to a control interface. It should beappreciated that the sensor assembly can include at least one sensor,e.g., a pressure sensor and/or temperature sensor and/or pH sensor, andat least two fluidic ports, arranged either as illustrated herein, butnot limited to the embodiments illustrated herein, as appreciated by theperson skilled in the art.

FIG. 7 illustrates another alternative placement of the fiber opticsensor assembly according to a fourth embodiment of the invention. InFIG. 7, the end of the sensor is positioned within a fluidic reservoiralong one of the fluidic ports. This embodiment provides the ability toutilize larger sensors that may be difficult to place within theconfines of the reservoir at the far tip of the holder. Examples of thefiber optic sensor assemblies are shown in FIG. 4B, wherein the fiberoptic sensor extends through the sample holder and eventually to acontrol interface. It should be appreciated that the sensor assembly caninclude at least one sensor, e.g., a pressure sensor and/or temperaturesensor and/or a pH sensor, and at least two fluidic pathways, arrangedeither as illustrated herein, but not limited to the embodimentsillustrated herein, as appreciated by the person skilled in the art. Anoptional third fluidic pathway is contemplated.

FIG. 8 illustrates the use of pressure or temperature sensors onnon-closed cell TEM holders. Electron Microscopes known as ETEMS(Environmental TEMs) establish a small localized gas environment at thesample area that is created by flowing gasses to and from the localizedsample area within an Electron Microscope. By placing a fiber opticpressure sensor within the localized gas region, the local pressure canbe measured. In the case of a sample heating holder, pressuredifferences at the sample will change the temperature of the sample. Thepressure value can be used by the system to compensate for this. Forexample, the pressure information could be used by the system todetermine an accurate temperature and/or be utilized to create a stabledetermined temperature by modifying the thermal input to the samplearea. Alternatively, a fiber optic temperature sensor can be mountedwithin the gas region for a direct measurement of the localizedtemperature.

Regardless of the embodiment, the sample holder described herein permitsthe user to ensure that the temperature, pressure and/or chemicalenvironment is maintained in real time at the precise requiredcondition.

Although the invention has been variously disclosed herein withreference to illustrative embodiments and features, it will beappreciated that the embodiments and features described hereinabove arenot intended to limit the invention, and that other variations,modifications and other embodiments will suggest themselves to those ofordinary skill in the art, based on the disclosure herein. The inventiontherefore is to be broadly construed, as encompassing all suchvariations, modifications and alternative embodiments within the spiritand scope of the claims hereafter set forth.

What is claimed is:
 1. An electron microscope sample holder comprising:a specimen tip defining a cell; and a fiber optic sensor assemblycomprising a fiber optic cable and a sensor end carried by the fiberoptic cable, the sensor end positioned in the specimen tip.
 2. Theelectron microscope sample holder according to claim 1, furthercomprising a barrel on which the specimen tip is mounted, wherein thefiber optic cable extends from the sensor end through the barrel.
 3. Theelectron microscope sample holder of claim 2, wherein at least onefluidic pathway in fluid communication with the cell extends along thebarrel.
 4. The electron microscope sample holder of claim 3, wherein theat least one fluidic pathway defines at least one of a fluid ingress anda fluid egress of the specimen tip.
 5. The electron microscope sampleholder of claim 4, wherein the sensor end of the fiber optic sensorassembly is positioned proximal to the at least one of a fluid ingressand a fluid egress of the cell.
 6. A sample support assembly for anelectron microscope sample holder specimen tip, the sample supportassembly comprising: a first sample support MEMS device having a firstthin membrane region; a second sample support MEMS device having asecond thin membrane region aligned with the first thin membrane regionwhen the sample support assembly is positioned in the specimen tip,wherein the first and second sample support MEMS devices areaccommodated within a fluidic reservoir.
 7. The sample support assemblyaccording to claim 6, wherein the second sample support MEMS device islarger than the first sample support device.
 8. The sample supportassembly according to claim 7, wherein the second sample support MEMSdevice overhangs beyond at least one side of the first sample supportMEMS device when the sample support MEMS devices are positioned in thespecimen tip.
 9. The sample support assembly according to claim 8,further comprising a first O-ring and a second O-ring.
 10. The samplesupport assembly according to claim 9, wherein, when the sample supportMEMS devices are positioned in the specimen tip, the first O-ringcontacts the first sample support MEMS device and the second O-ringcontacts the second sample support MEMS device.